There is recognition that gap junctions are important plasma membrane structures that help cells communicate with their environment. For example, most gap junctions are thought to assist passage of small molecules and ions between interconnected cells. Such movement is believed to exert profound effects on many aspects of cell physiology. Plasma membranes of adjacent cells are believed to include hemichannels, “connexons”, formed by multimeric proteins called “connexins” that help form the gap junctions. In addition, hemichannels play an independent role in the exchange of small molecular weight compounds between the cell cytoplasm and the periplasmic or extracellular space. See generally Bennett, M. et al. (1991) Neuron 6: 305; Kumar, N. and Gilula, N. B. (1996) Cell 84: 381; and Quist, A. P et al (2000) J. Cell Biol. 148: 1063 and references cited therein.
In particular, there have been reports that many gap junctions are specialized regions of the cell membrane with clusters of densely packed channels. Such gap junction channels are thought to directly connect the cytoplasmic compartment of two neighbouring cells.
There is recognition that gap junction channels are composed of two hemichannels (connexons) provided by each of two neighbouring cells. Each connexon (hemichannel) has been disclosed as consisting of six proteins called connexins. Each connexin is thought to share four transmembrane domains, two extracellular loops, and a cytoplasmic loop. The conduction of the electrical impulse is thought to take place through the gap junctions, thereby facilitating normal heart conduction and rhythm. See generally P. A. Guerrero, R. B. et al. J Clin Invest 1997, 99 1991; D. L. Lerner, K. A. et al., Circulation 1999, 99 1508; S. Kirchhoff, E. et al. Curr Biol 1998, 8 295.
Distribution of most heart connexins is thought to vary significantly throughout the organ. It has been disclosed that the Cx43 isoform is a major ventricular type while Cx40 is the most abundant isoform in the atrias and conductive system.
There are reports of strong links between connexin abnormalities and heart disease. See A. C. de Carvalho, et al., J Cardiovasc Electrophysiol 1994, 5 686; R. R. Kaprielian, et al., Circulation 1998, 97 651; N. S. Peters, et al., Circulation 1993, 88 864; and J. E. Saffitz, R. B. et al., Cardiovasc Res 1999, 42 309.
There is understanding that abnormal expression, distribution and regulation of gap junctions are involved in arrhythmias. The antiarrhythmic peptides disclosed by Larsen, B. et al. in PCT/DK01/00127 (WO01/62775) have been reported to increase gap junction intercellular communication (GJIC) in vertebrate tissue.
Particular mammalian gap junction proteins encoded by the connexin (Cx) gene family have been reported. See Bruzzone, R. et al. (1996) Eur. J. Biochem. 238: 1. The Cx family includes Cx26, 30, 31, 32, 37, 40, 43, 45, 46 and 50. Gap junction channels have also been found in invertebrates, where the channel forming proteins are called “innexins”
There is understanding that most connexins may be phosphorylated except for the Cx26 protein. The Cx43 protein is widely expressed in tissues. There are reports that phosphorylation of the Cx43 protein effects gap junction intracellular communication (GJIC). For example, there is acknowledgement that Cx43 turn over, trafficking, phosphorylation and gating are impacted by phosphorylation. See Darrow, B. J., et al. (1995). Circ Res 76: 381.
For example, Saffitz and co-workers have shown using conductance measurements that during ischemia there is an increase in connexin serine dephosphorylation within 15 minutes. See FIG. 1 (showing a mammalian Cx43 transmembrane protein with several identified phosphorylation sites).
The phosphorylation and solubility of the connexins has attracted research interest. In particular, Cx43 was found to be phosphorylated in the myoepithelial cells of rat mammary glands. See Wang, Y., et al. (1995). J Biol Chem 270, 26581; and Yamanaka, I., et al. (1997). Eur J Cell Biol 72: 166.
As mentioned, gap junction channels are thought to be specialized pores that connect the cytoplasm of neighboring cells. Hemichannels communicate with the extracellular environment. There have been reports that metabolic inhibition of heart cells can activate an influx pathway that may be structured by connexin hemichannels. Metabolic inhibition is thought to open the hemichannel and enhance loss of potassium, and induce influx of protons, sodium and calcium, thereby damaging heart tissue. See Kondo et al J. Mol. Cell. Cardiol. 32:1859–72, 2000; Li et al J. Mol. Cell. Cardiol. 33: 2145–55, 2001).
Electrical uncoupling at gap junctions during acute myocardial ischemia is believed to contribute to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca2+ and H+ and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling. Other mechanisms may play a role. For instance, it has been reported that uncoupling induced by acute ischemia is associated with changes in phosphorylation of connexin43 (Cx43). Results have been reported that are consistent with rapid, reversible Cx43 dephosphorylation playing a role in myocardial uncoupling and arrhythmogenesis during acute ischemia. See Beardslee M A et al., Circ Res. 2000;87:656 and references cited therein.
The structure and function of hemichannels have attracted interest.
For example, atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, have suggested that hemichannels are involved in extracellular calcium-dependent modulation of cell volume. As reported, cell volume changes were dependent on whether or not connexin43 was expressed. Changes were reported to be preventable by gap-junctional blockers (e.g., oleamide and beta-glycyrrhetinic acid) or were reversed by returning extracellular calcium to normal. It was suggested that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation. See Quist, A. P. et al, supra.
It has been proposed that open hemichannels, especially during ischemia or metabolic stress, may lead to cellular uptake of Ca2+, protons and accumulation of amphipathic lipid metabolites in cells causing cellular swelling, cell damage or apoptosis. See Beardslee et al., supra and Quist et al. supra.
There have been reports Cx43 is phosphorylated a positions Tyr247 (Y247), Tyr265 (Y265) and perhaps other positions by the activated Src protein in vitro. Significantly, gap junction intercellular communication (GJIC) was reported to be resistant to disruption by phosphorylation mediated by v-Src. It was acknowledged that phosphorylation on Y247 and Y265 of Cx43 is important. See Lin R, et al. (2001) J Cell Biol 154(4):815. See also FIG. 1.
See also Larsen, B. D et al. in a PCT application entitled New Medical Uses of Intracellular Communication Facilitating compounds as filed on 22 Feb. 2002 as PCT/US02/05773 (WO 02/077017) in which a variety of GJIC modulating compounds have been disclosed.
It would be desirable to have compounds that modulate hemichannel function. Preferred compounds would assist opening or closing of the hemichannel. It would be especially desirable to have molecular screens to identify such compounds.